The use of the ultracentrifuge to determine the catalytically competent forms of enzymes with more than one oligomeric structure. Multiple reacting forms of pyruvate carboxylase from chicken and rat liver.

The reacting enzyme sedimentation procedure has been used to identify the catalytically competent oligomeric forms of pyruvate carboxylases isolated from yeast and from chicken and rat liver. The latter two enzymes have been found to exist in at least two and three active oligomerit species, respectively. At low protein concentrations and with its substrates present, pyruvate carboxylase from chicken liver is a tetramer (sf,, = 16.4). However, when concentrated solutions of this enzyme are dialyzed against its substrates, a portion of the enzyme associates to a reacting form (s&,c = 22.7) which has been tentatively identified as an octamer. The activity of both forms is completely dependent on the presence of acetyl coenzyme A. The enzyme from chicken liver can also form monomers (SL,,,: = 7.51, particularly at low temperatures or alkaline pH, but no catalytic activity could be demonstrated for this form. In contrast to the chicken liver enzyme, pyruvate carboxylase from rat liver exists as an associating-dissociating mixture of tetramers (16 S), dimers (12 to 13 S), and monomers (6 to 7 S). All three oligomeric forms are catalytically active in the absence of acetyl-CoA although the latter compound strongly activates the dimer and probably affects the tetramer similarly. At low concentrations of salt and enzyme, the principal reacting form is the tetramer in the presence of acetyl-CoA, and the dimer in the absence of